Charcot-Marie-Tooth disease (CMT) is one of the most common inherited neurological disorders that affects ~ 1 in 2,500 people in the US. The disease is named for the three physicians who first identified it in 1886. CMT, also known as hereditary motor and sensory neuropathy (HMSN) or peroneal muscular atrophy, is made up of a group disorders that affect peripheral nerves. CMT affects both motor and sensory nerves. A typical characteristic includes weakness of the foot and lower leg muscles, which may result in foot drop and a high-stepped gait with frequent tripping or falls. Mutations in the small heat-shock protein 27 (HSPB1) cause axonal CMT or distal hereditary motor neuropathy (distal HMN). Our team has developed and characterized transgenic mice expressing mutant (S135F) HSPB1 in neurons only. These mice show all features of CMT2. Expression of mutant HSPB1 decreased acetylated -tubulin levels and induced severe axonal transport deficits. Of great significance, we have now shown that pharmacological inhibition of histone deacetylase 6 (HDAC6)-induced -tubulin deacetylation using our designed HDAC6I [HDAC6 Inhibitor] Tubastatin A corrected the axonal transport defects induced by HSPB1 mutations and rescued the CMT phenotype of symptomatic mutant HSPB1 mice. Our findings demonstrate the pathogenic role of -tubulin deacetylation in mutant HSPB1-induced neuropathies and offer valuable perspectives for HDAC6 inhibitors as a therapeutic strategy for hereditary axonopathies. In order to extend these high impact findings and to advance the best HDAC6I therapies to the clinic for the treatment of CMT and related neuropathies, we intend to more fully explore the structure activity relationships [SAR] of a family of Tubastatin A analogs, the key HDAC inhibitor used in our studies. The aims of this proposal are as follow: 1. Using the principles of medicinal chemistry and computer modeling methods, design and synthesize improved analogs of Tubastatin A and further optimize these for their in vitro HDAC isoform selectivity and potency. In this aim, in silico ADMET calculations will be performed so that only the most drug-like compounds are prepared for the preliminary in vitro studies. 2. For those compounds showing at least 300-fold selectivity for HDAC6 over the other isoforms and having an IC50 of < 50 nM, optimize these new chemical entities for their ability to enhance tubulin acetylation and mitochondrial movement in cultured neurons from symptomatic mutant HSPB1 mice. 3. For compounds showing the best effects in Aim 2, further characterize these compounds by determining their experimental ADMET parameters in order to ensure appropriate stability, cell permeability, lack of off- target activity, safety, etc. 4. Lastly, tes the best compounds from Aim 3 in the CMT transgenic mouse models, characterizing these as we have done before on their capacity to enhance tubulin acetylation in peripheral nerves and to reverse the symptoms in the mutant HSPB1 mice.